Estrogen receptor (ER) is a complex transcriptional regulatory factor containing discrete domains for hormone binding, DNA binding, and the transcriptional activation of responsive genes such as progesterone receptor. It has been hypothesized that loss of hormone dependence in breast tumors may sometimes be due to mutated or truncated ERs that activate transcription even in the absence of hormone. Based on our discovery of a transcriptionally active, hormone-independent ER variant in certain apparently ER-negative tumors, and other ER variants in ER-positive tumors, we suggest that there are a heterogeneous set of mutations within ER which might have important clinical implications. We therefore propose to search for sequence alterations within ER and PgR in specific tissues and tumor subpopulations, and to determine the effect of these alterations on receptor function. We will focus on four areas: 1) we will first study whether ER and PgR mRNA variants which we have recently observed have an altered function, using estrogen-responsive yeast and mammalian reporter systems; 2) we will search for ER mutants which have lost their hormone dependence and become independent stimulators of inappropriate breast tissue proliferation, using both ER/PgR-negative tumor specimens and existing proliferative breast lesions; 3) we will determine whether changes in transcription factors are responsible for the frequent loss of ER in large-cell comedo ductal carcinoma in situ, as compared to ER-expressing nonlarge-cell ductal carcinoma in situ; and 4) we will examine the molecular basis for the prognostically ominous loss of PgR during the time interval between primary tumor and metastasis, considering structural mutations in ER as well as PgR as possible causes, using primary/metastatic breast tumor pairs. These analyses will address central questions about ER and PgR structure and function, and provide insight into steroid receptor dysfunction in breast tumor evolution. Furthermore, these studies could have a direct impact on the use of ER and PgR in the management of breast cancer by providing a molecular basis for hormone unresponsiveness of independence.